The enteroviruses are a heterogeneous group of nearly 70 human pathogens which are responsible for a broad spectrum of clinical diseases. Like other members of the picornavirus family, the enteroviruses are small (27-nm), single-stranded, nonenveloped RNA viruses of approximately 1.34 g/ml buoyant density. Enteroviruses distinguish themselves from rhinoviruses, another type of human picornavirus, by their stability in acid, by their fecal-oral route of passage and transmission, and by their strict summer peak of disease activity. The prototypic enteroviruses, the polioviruses, remain the most clinically significant of the enteroviruses worldwide, causing paralytic disease in 4 of every 1,000 school-age children in developing countries. In the United States, the polioviruses have been controlled with the introduction of vaccines in the late 1950's. The nonpolio enteroviruses, however, are responsible for 5 to 10 million symptomatic infections each year. They are the most common etiologic agents of meningitis (75,000 cases per year) and of nonspecific febrile and exanthematous illnesses (5 million cases per year). They are also responsible for significant numbers of cases of myocarditis, hepatitis, pleurodynia, stomatitis, and neonatal sepsis. Recently identified nonpolio enterovirus serotypes cause hemorrhagic conjunctivitis and poliomyelitis mimicking that is due to the polioviruses. Several important diseases are suspected of having an enteroviral etiology without definitive proof; these include diabetes mellitus, dermatomyositis, congenital hydrocephalus, and amyotrophic lateral sclerosis. The enteroviruses cause infections which may persist for many years in immunocompromised individuals, often leading to death. Recently, a syndrome of late onset muscular atrophy has been reported in individuals who suffered paralytic poliomyelitis 20 to 40 years previously.
Beyond the obvious desire to determine the specific etiology of these diverse and important diseases, there are many reasons for seeking a rapid and accurate diagnostic test for the enteroviruses. It is often clinically impossible to distinguish enteroviral infections from those due to bacterial pathogens or other viruses, including herpes simplex, for which there are specific therapies. Although many enteroviral infections are self-limited and require no therapy, the fear that an illness may be bacterial or herpetic results in unnecessary hospitalization and antibiotic or antiviral treatment for thousands of enterovirus-infected patients each year. Certain enteroviral diseases are in fact severe enough to warrant specific therapy, were such available. Indeed, several experimental agents have been shown to be very effective against the enteroviruses in vitro and in animal models. These have never been studied in humans, however, because the diagnosis of enterovirus infection is currently made too slowly to conduct an appropriate clinical drug trial. Finally, as alluded to earlier, a number of diseases are theorized to be due to the enteroviruses but have not yet been proven as such. A clear association with these viruses would facilitate the understanding and treatment of such conditions.
The Nobel Prize in medicine and physiology was awarded to J. F. Enders, F. C. Robbins, and T. H. Weller in 1954 for their success in cultivating poliovirus in tissue culture, an accomplishment which paved the way for vaccine development and provided a means for laboratory testing for the polio and nonpolio enteroviruses. Since then, tissue culture continues to be the mainstay of the enteroviral diagnosis despite well-recognized limitations. Tissue culture is time-consuming and requires a high level of expertise. Of greater concern is the fact that certain of the enteroviruses will not grow in tissue culture, requiring inoculations into suckling mice for detection, a technique cumbersome enough to be omitted from almost all diagnostic laboratories. The sensitivity of routine tissue culture for the enteroviruses may be as low as 65 to 75%, and development of characteristic cytopathic effect may take too long to be of benefit to the patient. Cerebrospinal fluid (CSF) infections with the enteroviruses take a mean of 6.3 days in the laboratory for growth in culture, consistent with reported means to isolation from the CSF of 4.0 to 8.2 days. Other body sites may become positive sooner, but as meningitis is the most vexing of enteroviral infections for the clinician, CSF data are the most relevant. The use of additional cell lines improves the yield at the cost of increasing the labor and resource required.
Immunodiagnostic techniques for the enteroviruses have been fraught with difficulties resulting from the extreme antigenic diversity among the serotypes. Although a common antigen may exist among the polioviruses and another among the coxsackievirus B types, checkerboard pools of antisera would be required to cover even the most common enteroviral serotypes responsible for human disease. Serologic testing suffers from the same lack of a ubiquitous enteroviral antigen as immunoassays do, requiring, in this case, pools of antigens for testing. Coxsackievirus type B immunoglobulin M serology has the most proven clinical application. It has been found to be advantageous because of shared antigen and early appearance of the immunoglobulin M class of antibodies. Immunoglobulin G serology for the enteroviruses is useful for epidemiologic studies, but of little benefit to the individual patient.
DNA and RNA probes have been used to detect enteroviruses. In Rotbart et al., J. Clin. Microbiol. 20: 1105-1108, (1984), three nucleotide hybridization probes derived from DNA clones of the poliovirus type 1 genome were used in dot hybridization experiments. The probes successfully detected members of each of the major enteroviral subgroups. In Rotbart et al., J. Clin. Microbiol 22: 220-224, (1985), cDNA probes derived from poliovirus 1 and coxsackievirus B3 were used to detect enteroviruses in cerebrospinal fluid reconstruction experiments where an array of enteroviruses were added to cerebrospinal fluid. The viruses were detected by a dot hybridization assay using cDNA probes. Although cDNA probes have been able to detect enteroviruses in cerebrospinal fluid reconstructions, in clinical tests the probes were relatively insensitive in detecting enteroviral infection, Rotbart and Levin, Chapter 15, "Progress Toward the Development of a Pan-Enteroviral Nucleic Acid Probe", in DNA Probes for Infectious Diseases, pp. 193-209, 197. In the clinical tests, two thirds of cerebrospinal test fluids that proved positive with tissue culture were missed by the cDNA probes. Single stranded RNA probes (Rotbart et al., Molecular and Cellular Probes 2: 65-73, (1988) can be several times more sensitive than cDNA probes, however, even this improved sensitivity may be too little to routinely detect enteroviruses in cerebrospinal fluid. It is estimated that cerebrospinal fluid from patients with aseptic meningitis due to human enterovirus contains 10.sup.1 -10.sup.3 virions per milliliter. The sensitivity of the RNA probes approached this level; nevertheless, the low levels of virus in body fluids preclude the reliable use of the probes for diagnosing picornaviral infection on a routine basis.
There is thus a great need for sensitive methods for detecting picornaviruses in biological fluids and tissues that can be applied to the small amounts of virus often present and that can be quickly performed so that timely diagnosis of infection can be made.